New Paper - Gammaretroviruses - Maureen Hanson, David Bell

Thanks for the analysis from those that have read the full paper, esp Mark.

This looked interesting to me:

In addition, 5 additional samples irreproducibly gave gagO/gagI PCR products and 11 sometimes were PCR positive for gagL; due to the inconsistency, we decided to score these as negative.

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IIRC, Judy Mikovits said they scored inconsistently reproducible results as a positive. I wonder how much this different approach to scoring could account for the difference between the Science results and these latest ones? [excluding the retracted Silverman results from the Science ones].

IIRC, Judy Mikovits said they scored inconsistently reproducible results as a positive. I wonder how much this different approach to scoring could account for the difference between the Science results and these latest ones? [excluding the retracted Silverman results from the Science ones].

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Yes, that caught my eye too Oceanblue. Particularly interesting in relation to the PCR inhibition problem identified by O'Keefe, and as you say, Mikovits also reported that for some samples, several rounds of testing were sometimes required in order to get a few inconsistent positives and conclude that the sample was positive.

Those observations of Mikovits probably seemed very dubious to sceptical commentators, leading them to suggest extreme and unrealistic scenarios where Mikovits had just lost all perspective and tested the patient samples again and again until they were found positive, while not doing the same for healthy control samples. Those kinds of suggestions always seemed to me to be utterly unrealistic, insulting and disrespectful of an experienced and highly competent expert in this field, and developments in the last year, including this observation in this study, indicate that all of these very difficult problems that are being wrestled with really are yielding genuinely groundbreaking developments in the understanding of PCR. And O'Keefe's work, apparently confirmed in this study too, suggests that Mikovits may well have been right about this point...and ahead of her time...

O'Keefe's finding may well prove to be the beginning of the most significant development of all in those entire story, in the long term, because if the new science that is being uncovered here reveals that PCR has weaknesses both in false positives (previously unknown effects of previously unknown widespread forms of contamination) and false negatives (contamination from the test itself which affects subsequent tests, and perhaps further unknown reasons that cause false negatives), then this better understanding of PCR would have widespread implications beyond retrovirology.

As I've said many times before, similar anomalous results that have not proved reproducible have been discarded for decades; problems introduced with many rounds of PCR remain unexplained and researchers differ on how many rounds are too many - but throwing those anomalies in the bin because you can't reproduce them is just not good enough. One should be curious about such matters; a curious scientist should want to get to the bottom of this question rather than dismissing such results with unproven explanations. Both in the laboratory and in the surgery, far too many unexplained anomalous results are discarded and forgotten, thrown into a wastebasket as rubbish rather than treated as a rich source of clues about the frontiers of knowledge. As patients, we understand this process all too well, our illness has been fundamentally defined out of existence on the medical map by systematising such a rubbish bin. It seems ironic, but may perhaps be inevitable, that the scientific solution to our problems may end up with us digging up various scraps of discarded paper from past research and testing, and saying "wow! now we finally understand what all those strange anomalies were about!"

Good point Mark. Scientists should be curious about things that "dont fit" They are clues to the unknown.
That is why I have been so dismayed by the energy with which Dr Mikovits findings have been ridiculed and dismissed.
I know that not all researchers have responded in this way, but many of the most prominent seem to have been guilty of it.

There is quite a history of new retroviruses having been initially associated with a disease, only to experience the same problem of reproducibility with PCR testing we have seen with "XMRV"
All these early disease associations from the 1970s and 80s have been discarded.

I seem to remember that there were viral associations made in leukaemia which could never be reliably reproduced once researchers came to rely exclusively on PCR to validate their initial findings. Could be that PCR is misleading in some unpredictable and unsuspected way and these old viral associations, now discarded, could be correct?

I remember an early talk in which Mikovits remarked that the present generation of retrovirologists have been trained to rely too exclusively on PCR findings, and contrasted that with earlier approaches taken by "classically trained" virologists - before they could just rely on the technology.

Mikovits used to score the inconsistent positives as positive because she said it depended on the life cycle of the retrovirus,which had to be caught at a point when it was not locked up in the DNA.

Yes, that caught my eye too Oceanblue. Particularly interesting in relation to the PCR inhibition problem identified by O'Keefe, and as you say, Mikovits also reported that for some samples, several rounds of testing were sometimes required in order to get a few inconsistent positives and conclude that the sample was positive.

Those observations of Mikovits probably seemed very dubious to sceptical commentators, leading them to suggest extreme and unrealistic scenarios where Mikovits had just lost all perspective and tested the patient samples again and again until they were found positive, while not doing the same for healthy control samples. Those kinds of suggestions always seemed to me to be utterly unrealistic, insulting and disrespectful of an experienced and highly competent expert in this field, and developments in the last year, including this observation in this study, indicate that all of these very difficult problems that are being wrestled with really are yielding genuinely groundbreaking developments in the understanding of PCR. And O'Keefe's work, apparently confirmed in this study too, suggests that Mikovits may well have been right about this point...and ahead of her time...

O'Keefe's finding may well prove to be the beginning of the most significant development of all in those entire story, in the long term, because if the new science that is being uncovered here reveals that PCR has weaknesses both in false positives (previously unknown effects of previously unknown widespread forms of contamination) and false negatives (contamination from the test itself which affects subsequent tests, and perhaps further unknown reasons that cause false negatives), then this better understanding of PCR would have widespread implications beyond retrovirology.

As I've said many times before, similar anomalous results that have not proved reproducible have been discarded for decades; problems introduced with many rounds of PCR remain unexplained and researchers differ on how many rounds are too many - but throwing those anomalies in the bin because you can't reproduce them is just not good enough. One should be curious about such matters; a curious scientist should want to get to the bottom of this question rather than dismissing such results with unproven explanations. Both in the laboratory and in the surgery, far too many unexplained anomalous results are discarded and forgotten, thrown into a wastebasket as rubbish rather than treated as a rich source of clues about the frontiers of knowledge. As patients, we understand this process all too well, our illness has been fundamentally defined out of existence on the medical map by systematising such a rubbish bin. It seems ironic, but may perhaps be inevitable, that the scientific solution to our problems may end up with us digging up various scraps of discarded paper from past research and testing, and saying "wow! now we finally understand what all those strange anomalies were about!"

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Hi Mark

I'm not sure that JM repeatedly testing her samples to get a few positives is fully consistent with the O'Keefe finding: it sounds like JM had negatives on the first test and positives on later tests too - rather than lots of early positives that couldn't be replicated (possbily because of inhibiting contamination). Maybe someone has more information on this. And does anyone know if JM explicitly confirmed that controls were tested as many times as patients? That was exactly what concerned me when she first made the comment but never saw any clarification. This isn't a question of insulting this or that scientist but trying to fully understand the science. Maybe JM did clarify this but if so I missed it (it wasn't easy to keep up with the XMRV maelstrom). Thanks

There is quite a history of new retroviruses having been initially associated with a disease, only to experience the same problem of reproducibility with PCR testing we have seen with "XMRV"
All these early disease associations from the 1970s and 80s have been discarded.
.

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That's actually a large part of the reason for a lot of the scepticism and disbelief, and the efforts to disprove the results quickly and move on. It was explicitly evident from comments on (eg) Bad Science that a lot of people just said "oh no, another virus scare, another pointless waster of money on a fruitless search". It's become kind of assumed that results like this won't pan out, based on so many such failures in the past. Obviously that's a problem for dispassionate and objective enquiry, but also it does strongly suggest that there has been something unknown going own. Sceptics would still say "yes: contamination", and they may be largely right, but also it is now fairly clear that there are issues with PCR; as you rightly say, it's actually a young and convenient technology, rather than a mature and completely reliable one.

Just thinking about the O'Keefe paper. I don't think it should apply to successive testing if changing to a different type of PCR test. I think it only applies to successive testing using the same PCR slides, or to any testing kits that have been previously contaminated. In other words, it's the slides that get contaminated, and not the blood samples. A fresh slide is not automatically exempt from contamination, because the contaminating product can be used before the experiment for calibration. Therefore successive tests using the same PCR test might be affected, but so might the first test. But when switching to a new type of test, the chances for contamination start afresh.

That's going from memory and a bit of guess-work, but without re-reading the O'Keefe paper, I can't remember the exact details.

Alex, we can just do the other studies with no spinal injections???
I think the one they are really chasing first is testing nk function and comparing cfs to rheumatoid arthritis and ms. Its sounds like they have a plan for alot more in the future.

I've managed to run my eye over it now, but I've not read it carefully yet, and so I may have made some mistakes, but here are some extracted titbits:

Study Subjects:

David Bell provided 10 severely ill patient samples (Fukuda), 10 'recovered'* patients, and 20 control samples.
Susan Levine provided samples from 20 CFS patients and 4 healthy controls.
12 controls who have never been diagnosed with CFS were recruited from Ithaca, New York.

So that's 30 patients, 10 'recovered'* patients and 36 controls, if i've added those up correctly.

* The 'recovered' patients, once fitted the Fukuda criteria but now consider themselves recovered.

A round-up of the positive results:

MLV-like gag sequences were obtained following PCR of gDNA and cDNA from PBMCs from subjects in Western New York:

"Taken together, 5 samples from severe patients, 2 from recovered CFS patients, and 3 from control subjects resulted in detection of gag PCR products. All of these samples were negative in mouse mtDNA assays. All PCR results were confirmed by sequencing."

So that's 7 positives from patients, and 3 positives from controls.

MLV-like gag sequences detected in LNCaP cultures:

"Two samples gave gagO/gagI PCR products: 11F2-P4 and 14F2-P4. With gagL PCR, 7 samples were positive: 2F1-P6, 4F1-P6, 9F1-P6, 10F3-P6, 11F2-P4, 12F2-P6, and 13F3-P6. In addition, 5 additional samples irreproducibly gave gagO/gagI PCR products and 11 sometimes were PCR positive for gagL; due to the inconsistency, we decided to score these as negative."

So that's at least 18 that were positive for gagL, and 7 positive for gagO, out of 40 subjects for this test. (I assume that the '40 subjects' breaks down as: the 30 patients, and the 10 'recovered' patients.)
It doesn't give numbers for the controls as far as i can see, and it doesn't give a break-down for the 30 patient samples and the 10 'recovered' patient samples.
And it doesn't give a total for how many were positive for any sequences, as far as I can see.

gag PCR of DNA from PBMCs of[12] control subjects in Ithaca, New York:

"We observed a gagL PCR fragment only one time in an Ithaca control sample"

Phylogenetic Trees:

The phylogenetic trees show that the sequences were closer to Lo's PMRVs than to XMRVs, and were distinct from 22Rv1. (I'm not yet certain if this applies to all of the sequences.)

"The trees illustrate that the sequences amplified from the samples of this study were related to polytropic MLV-like sequences, and that all gagL sequences were distinct from 22Rv1 XMRV, which contains deletions in the gag leader region."

"There are differences among the sequences we obtained and between our sequences and those of Lo et al. [9]; however, it should be noted that relatively small sequences, approximately 400 nt, are being compared."

Some interesting quotes:

"Whether a retrovirus is involved in inciting or maintaining CFS/ME will require further investigation using other types of assays."

"Like a number of other investigators, we have identified MLV-like gag sequences following PCR of nucleic acid samples from whole blood or PBMCs from human subjects. Nested PCR analysis of our initial batch of 30 samples resulted in a significant difference in frequency of gag PCR products between patients and controls; however, continued analysis failed to maintain this association."

MLV-like gag sequences were obtained following PCR of gDNA and cDNA from PBMCs from subjects in Western New York:"Taken together, 5 samples from severe patients, 2 from recovered CFS patients, and 3 from control subjects resulted in detection of gag PCR products. All of these samples were negative in mouse mtDNA assays. All PCR results were confirmed by sequencing."
So that's 7 positives from patients, and 3 positives from controls.

MLV-like gag sequences detected in LNCaP cultures:"Two samples gave gagO/gagI PCR products: 11F2-P4 and 14F2-P4. With gagL PCR, 7 samples were positive: 2F1-P6, 4F1-P6, 9F1-P6, 10F3-P6, 11F2-P4, 12F2-P6, and 13F3-P6. In addition, 5 additional samples irreproducibly gave gagO/gagI PCR products and 11 sometimes were PCR positive for gagL; due to the inconsistency, we decided to score these as negative."

So that's at least 18 that were positive for gagL, and 7 positive for gagO, out of 40 subjects for this test.
It doesn't give numbers for the controls as far as i can see.
And it doesn't give a total for how many were positive for any sequences, as far as I can see.

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Thanks for all this, very helpful.

The LNCaP results look like 8 consistent positives (2 for GagO, 7 for gagL with 11F2-P4 positive for both), plus 16 more that were inconsistently positive and so scored negative.Total 24 positive at least some of the time ex 40 patients?

If I've got that right (not sure I have) that's an interesting finding both for the number of confirmed positives - and the inconsistent positives that Hansen scored negative, but presumably the original Science paper would have scored positive. 24/40=60% 'positives' which I think tallies broadly with the Science paper.

The LNCaP results look like 8 consistent positives (2 for GagO, 7 for gagL with 11F2-P4 positive for both), plus 16 more that were inconsistently positive and so scored negative.Total 24 positive at least some of the time ex 40 patients?

If I've got that right (not sure I have) that's an interesting finding both for the number of confirmed positives - and the inconsistent positives that Hansen scored negative, but presumably the original Science paper would have scored positive. 24/40=60% 'positives' which I think tallies broadly with the Science paper.

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Yes, it looks like 8 in total were positive, and then the additional samples were 'irreproducibly' positive: 5 samples gave gagO/gagI PCR products, and 11 gave gagL PCR products. But does this mean there were 16 additional samples, or was there an overlap between gagO and gagL? It isn't clear.

So in total, it could be between 19 (8+11) and 24 (8+16) samples that were positive, out of 40 patient samples, 30 of which had a current diagnosis and 10 of which had 'recovered'.

19 would be 47% positive
24 would be 60% positive

But this doesn't take into account the 7 positives from the other tests (PCR of gDNA and cDNA from PBMCs), which might have been additional positive samples. I don't think they've included enough data for us to work out the total positives in either the patient samples or the control samples. I haven't found the info yet anyway. If those 7 positives were additional, then it could be a total of up to 31 positive patient samples out of 40 (30 current patients, and 10 'recovered'), which would be 77%.

I can't find many figures for the controls, beyond the 3 in the 'PCR of gDNA and cDNA from PBMCs' tests, so I can't work out the total for controls.

Thanks, Bob. You're right, the range is 47%-60% positive, and that excludes positives from the non-LNCap tests. Would have been nice if they'd given tallies for positives/sometimes-positives across all tests, but from the data given it does look like their results broadly tally with the original Science paper if intermittent positives are scored 'positive' rather than 'negative', which makes this a very interesting paper indeed.

Thanks, Bob. You're right, the range is 47%-60% positive, and that excludes positives from the non-LNCap tests. Would have been nice if they'd given tallies for positives/sometimes-positives across all tests, but from the data given it does look like their results broadly tally with the original Science paper if intermittent positives are scored 'positive' rather than 'negative', which makes this a very interesting paper indeed.

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Yes, it is very interesting, isn't it. It is actually a positive replication study, or sorts, of both the Mikovits and the Lo papers.
But they have no ideas why they are getting these results. From what I understand, so far, this study completely vindicates Judy Mikovits, and confirms her research. The only difference is the interpretation.

What gives me hope, about the science in general, is that this Hanson paper has been completely open and honest about publishing their exact results, with no obfuscation, and no over-blown conclusions. Nothing is hidden, so the information is out there for others to follow up.
And people like Denise O'Keefe are clearly interested in seeing this research followed up.
Maybe we are at a stage with technology now, where these results may be understood over the next couple of years.

Apart from anything else, they are finding unexplained MLV-like sequences which are not mouse contamination, as far as they can determine, and are not 22RV1. So what are these sequences, and where do they originate? No one seems to have any idea at all. If it isn't mouse contamination, then what is it?

(I haven't scrutinised the sequences yet, and so I don't know how similar they are to mouse ERV sequences - Has anyone else had a look yet?)

The BPH viruses disclosed herein are related to previously identified gammaretroviruses such as murine leukemia virus and xenotropic murine leukemia virus-related virus (XMRV), but are distinct from these viruses based on nucleic acid and amino acid sequences. Therefore, the methods disclosed herein include methods of specifically detecting BPH virus (such as a BPH virus polynucleotide or polypeptide) in a sample from a subject, for example discriminating the presence of BPH virus in a sample as opposed to XMRV. In some examples, the disclosed methods include detecting the presence of a BPH virus and the absence of XMRV in a sample from a subject.

Yes, it is very interesting, isn't it. It is actually a positive replication study, or sorts, of both the Mikovits and the Lo papers.
But they have no ideas why they are getting these results. From what I understand, this study completely vindicates Judy Mikovits, and confirms her research. The only difference is the interpretation.

...Apart from anything else, they are finding unexplained MLV-like sequences which are not mouse contamination, as far as they can determine, and are not 22RV1. So what are these sequences, and where do they originate? No one seems to have any idea at all. If it isn't mouse contamination, then what is it?
(I haven't scrutinised the sequences yet, and so I don't know how similar they are to mouse ERV sequences - Has anyone else had a look yet?)

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Hi Bob

Yes, these results are fascinating and in line with the original Science resutls. However, I don't think they wholly exonerate Judy Mikovits as declaring inconsistent results as positives is a questionable practice, particularly as this wasn't mentioned within the original Science paper.

As you say, the other really interesting aspect is that Hanson found the sequenced MLV's were closer to Lo's than XMRV clones. JM also said she was finding non-XMRV but MLV-related sequences, though I don't think this was actually published.

I don't know how this new paper's results square with the definitive negative papers published before, but it does seem to rule out a simple contamination explanation.

Maybe we still need to wait for the Lipkin study to see how this all pans out.

Yes, these results are fascinating and in line with the original Science resutls. However, I don't think they wholly exonerate Judy Mikovits as declaring inconsistent results as positives is a questionable practice, particularly as this wasn't mentioned within the original Science paper.

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I thought she had declared that she got inconsistent results. It was widely known that she had to test individual samples multiple times, and if she got one positive, then she would mark that up as a positive sample. But I can't remember how much of that was actually published in the original paper. Is that questionable practise if you are clear and transparent about your methodology?

In any case, it seems to be a difference of interpretation, rather than a difference in the actual results. Would you agree?

As you say, the other really interesting aspect is that Hanson found the sequenced MLV's were closer to Lo's than XMRV clones. JM also said she was finding non-XMRV but MLV-related sequences, though I don't think this was actually published.